LOCUS       sequence-246554-        6477 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses UBE2Q2 with N-terminal Strep-HA tag in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6477)
  AUTHORS   Koerver L, Papadopoulos C, Liu B, Kravic B, Rota G, Brecht L, 
            Veenendaal T, Polajnar M, Bluemke A, Ehrmann M, Klumperman J, 
            Jaattela M, Behrends C, Meyer H
  TITLE     The ubiquitin-conjugating enzyme UBE2QL1 coordinates lysophagy in 
            response to endolysosomal damage.
  JOURNAL   EMBO Rep. 2019 Aug 21:e48014. doi: 10.15252/embr.201948014.
  PUBMED    31432621
REFERENCE   2  (bases 1 to 6477)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "EMBO Rep. 
            2019 Aug 21:e48014. doi: 10.15252/embr.201948014."
COMMENT     Sequence Label: pcDNA5FRT/TO-Strep/HA-UBE2Q2
FEATURES             Location/Qualifiers
     source          1..6477
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        109..488
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        489..692
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     643..663
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    694..712
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    715..733
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     743..767
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             938..964
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             995..1018
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1055..1078
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     protein_bind    1091..1115
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2241..2265)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2303..2320)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2309..2533
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2666..2685)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2817..2864
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2872..3894
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2872..3894
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4024..4145
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4061..4080)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4115..4134
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4194..4210)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4194..4210)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4207..4229)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4218..4234
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4242..4248,4249..4266,4267..4272))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4287..4308
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4425..4442)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4596..5184)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4676..4695)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5355..6146,6147..6215))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5978..5997
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6216..6320)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6395..6414)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg
       61 ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat
      121 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      181 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      241 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      301 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      361 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      421 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      481 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      541 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      601 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      661 gtgtacggtg ggaggtctat ataagcagag ctctccctat cagtgataga gatctcccta
      721 tcagtgatag agatcgtcga cgagctcgtt tagtgaaccg tcagatcgcc tggagacgcc
      781 atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc cggactctag
      841 cgtttaaact taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg
      901 cagatatcca gcacagtggc ggccgctcga gaccatgtac ccatacgatg ttcccgacta
      961 cgccggtacc gagctcggat ccaccatggc tagctggagc cacccgcagt tcgagaaagg
     1021 tggaggttcc ggaggtggat cgggaggtgg atcgtggagc cacccgcagt tcgaaaaagc
     1081 ggccgatatc acaagtttgt acaaaaaagt tggcatgtcc gtgtcagggc tcaaggccga
     1141 gctgaagttc ctggcgtcca tcttcgacaa gaaccacgag cgattccgca tcgtcagttg
     1201 gaagctggac gagctgcact gccagttcct ggtgccgcag cagggcagcc cgcactcgct
     1261 gccgccgcca ctcacgctcc actgcaacat cacggaatcc tatccatctt cttcaccgat
     1321 atggtttgtg gattctgaag acccaaatct gacatcagtt ctggaacgtc tagaagatac
     1381 taagaacaac aatttgcttc gtcagcaatt gaagtggttg atatgtgaac tctgcagttt
     1441 atataacctt cctaagcacc tggatgttga gatgctagat caaccactac ccacgggtca
     1501 gaatgggaca acagaagaag tgacttcaga agaagaggaa gaagaagaag agatggctga
     1561 agatatagaa gacttagatc actatgagat gaaggaagaa gagcctatta gtgggaaaaa
     1621 gtcagaggat gaaggaattg aaaaagaaaa tttggcaata ttagagaaaa ttaggaagac
     1681 tcaaaggcaa gaccatttaa atggtgcagt gtctgggtca gtgcaagctt cagatagact
     1741 tatgaaagag ctcagggaca tatacagatc acagagttat aaaacaggga tttattcagt
     1801 ggaactcata aatgacagtt tatatgactg gcatgttaaa ctgcagaagg ttgaccctga
     1861 tagtcctttg cacagtgatc ttcagatctt aaaagaaaaa gaaggcatag aatatatttt
     1921 gcttaacttc tcttttaagg ataactttcc atttgatcct ccatttgttc gagtggtgtt
     1981 acctgttctc tcaggagggt atgtattggg tggaggagca ttatgtatgg aacttctcac
     2041 aaaacagggc tggagcagtg cctactcaat agaatcggtc atcatgcaaa taaatgccac
     2101 cttagtcaaa ggcaaagcca gagtgcagtt tggagcaaat aagaatcaat ataatctagc
     2161 aagagcccaa caatcctata attccattgt acagatacat gagaaaaatg gctggtacac
     2221 ccctccaaag gaagatggct acccaacttt cttgtacaaa gtggtgacgt aagctagggg
     2281 cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt
     2341 tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa
     2401 taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg
     2461 gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg
     2521 gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg gtatccccac
     2581 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     2641 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     2701 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     2761 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tacctagaag
     2821 ttcctattcc gaagttccta ttctctagaa agtataggaa cttccttggc caaaaagcct
     2881 gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag cgtctccgac
     2941 ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt aggagggcgt
     3001 ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg ttatgtttat
     3061 cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg ggaattcagc
     3121 gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca agacctgcct
     3181 gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg
     3241 gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat cggtcaatac
     3301 actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca ctggcaaact
     3361 gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg
     3421 gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc caacaatgtc
     3481 ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat gttcggggat
     3541 tcccaatacg aggtcgccaa catcttcttc tggaggccgt ggttggcttg tatggagcag
     3601 cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg gctccgggcg
     3661 tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg caatttcgat
     3721 gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc
     3781 gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg tgtagaagta
     3841 ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga atagcacgta
     3901 ctacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     3961 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     4021 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4081 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4141 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     4201 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4261 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4321 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4381 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4441 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4501 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4561 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4621 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4681 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4741 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4801 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4861 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4921 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4981 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     5041 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5101 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5161 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5221 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5281 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5341 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5401 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5461 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5521 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5581 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5641 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5701 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5761 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5821 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5881 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5941 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     6001 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6061 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6121 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6181 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6241 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6301 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     6361 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6421 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagca
//
